Enhancement of the d-Allulose 3-Epimerase Expression in Bacillus subtilis through Both Transcriptional and Translational Regulations.

d-Allulose, a functional bulk sweetener, has recently attracted increasing attention because of its low-caloric-ness properties and diverse health effects. d-Allulose is industrially produced by the enzymatic epimerization of d-fructose, which is catalyzed by ketose 3-epimerase (KEase). In this study, the food-grade expression of KEase was studied using Bacillus subtills as the host. Clostridium sp. d-allulose 3-epimerase (Clsp-DAEase) was screened from nine d-allulose-producing KEases, showing better potential for expression in B. subtills WB600. Promoter-based transcriptional regulation and N-terminal coding sequence (NCS)-based translational regulation were studied to enhance the DAEase expression level. In addition, the synergistic effect of promoter and NCS on the Clsp-DAEase expression was studied. Finally, the strain with the combination of a PHapII promoter and gln A-Up NCS was selected as the best Clsp-DAEase-producing strain. It efficiently produced Clsp-DAEase with a total activity of 333.2 and 1860.6 U/mL by shake-flask and fed-batch cultivations, respectively.

Enhancing enzyme immobilization: Fabrication of biosilica-based organic-inorganic composite carriers for efficient covalent binding of D-allulose 3-epimerase.

D-allulose, an ideal low-calorie sweetener, is primarily produced through the isomerization of d-fructose using D-allulose 3-epimerase (DAE; EC 5.1.3.30). Addressing the gap in available immobilized DAE enzymes for scalable commercial D-allulose production, three core-shell structured organic-inorganic composite silica-based carriers were designed for efficient covalent immobilization of DAE. Natural inorganic diatomite was used as the core, while 3-aminopropyltriethoxysilane (APTES), polyethyleneimine (PEI), and chitosan organic layers were coated as the shells, respectively. These tailored carriers successfully formed robust covalent bonds with DAE enzyme conjugates, cross-linked via glutaraldehyde, and demonstrated enzyme activities of 372 U/g, 1198 U/g, and 381 U/g, respectively. These immobilized enzymes exhibited an expanded pH tolerance and improved thermal stability compared to free DAE. Particularly, the modified diatomite with PEI exhibited a higher density of binding sites than the other carriers and the PEI-coated immobilized DAE enzyme retained 70.4 % of its relative enzyme activity after ten cycles of reuse. This study provides a promising method for DAE immobilization, underscoring the potential of using biosilica-based organic-inorganic composite carriers for the development of robust enzyme systems, thereby advancing the production of value-added food ingredients like D-allulose.

A prototrophic suppressor of a Vibrio fischeri D-glutamate auxotroph reveals a member of the periplasmic broad-spectrum racemase family (BsrF).

Although bacterial peptidoglycan (PG) is highly conserved, some natural variations in PG biosynthesis and structure have evolved. Understanding the mechanisms and limits of such variation will inform our understanding of antibiotic resistance, innate immunity, and the evolution of bacteria. We have explored the constraints on PG evolution by blocking essential steps in PG biosynthesis in Vibrio fischeri and then selecting mutants with restored prototrophy. Here, we attempted to select prototrophic suppressors of a D-glutamate auxotrophic murI racD mutant. No suppressors were isolated on unsupplemented lysogeny broth salts (LBS), despite plating >1011 cells, nor were any suppressors generated through mutagenesis with ethyl methanesulfonate. A single suppressor was isolated on LBS supplemented with iso-D-gln, although the iso-D-gln subsequently appeared irrelevant. This suppressor has a genomic amplification formed by the creation of a novel junction that fuses proB to a gene encoding a putative broad-spectrum racemase of V. fischeri, bsrF. An engineered bsrF allele lacking the putative secretion signal (ΔSS-bsrF) also suppressed D-glu auxotrophy, resulting in PG that was indistinguishable from the wild type. The ΔSS-bsrF allele similarly suppressed the D-alanine auxotrophy of an alr mutant and restored prototrophy to a murI alr double mutant auxotrophic for both D-ala and D-glu. The ΔSS-bsrF allele increased resistance to D-cycloserine but had no effect on sensitivity to PG-targeting antibiotics penicillin, ampicillin, or vancomycin. Our work helps define constraints on PG evolution and reveals a periplasmic broad-spectrum racemase in V. fischeri that can be co-opted for PG biosynthesis, with concomitant D-cycloserine resistance. IMPORTANCE: D-Amino acids are used and produced by organisms across all domains of life, but often, their origins and roles are not well understood. In bacteria, D-ala and D-glu are structural components of the canonical peptidoglycan cell wall and are generated by dedicated racemases Alr and MurI, respectively. The more recent discovery of additional bacterial racemases is broadening our view and deepening our understanding of D-amino acid metabolism. Here, while exploring alternative PG biosynthetic pathways in Vibrio fischeri, we unexpectedly shed light on an unusual racemase, BsrF. Our results illustrate a novel mechanism for the evolution of antibiotic resistance and provide a new avenue for exploring the roles of non-canonical racemases and D-amino acids in bacteria.

Interrogating l-fuconate dehydratase with tartronate and 3-hydroxypyruvate reveals subtle differences within the mandelate racemase-subgroup of the enolase superfamily.

Enzymes of the enolase superfamily share a conserved structure and a common partial reaction (i.e., metal-assisted, Brønsted base-catalyzed enol(ate) formation). The architectures of the enolization apparatus at the active sites of the mandelate racemase (MR)-subgroup members MR and l-fuconate dehydratase (FucD) are almost indistinguishable at the structural level. Tartronate and 3-hydroxypyruvate (3-HP) recognize the enolization apparatus and can be used to interrogate the active sites for differences that may not be apparent from structural data. We report a circular dichroism-based assay of FucD activity that monitors the change in ellipticity at 216 nm (Δ[Θ]S-P = 8985 ± 87 deg cm2 mol-1) accompanying the conversion of l-fuconate to 2-keto-3-deoxy-l-fuconate. Tartronate was a linear mixed-type inhibitor of FucD (Ki = 8.4 ± 0.7 mM, αKi = 63 ± 11 mM), binding 18-fold weaker than l-fuconate, compared with 2-fold weaker binding of tartronate by MR relative to mandelate. 3-HP irreversibly inactivated FucD (kinact/KI = 0.018 ± 0.002 M-1s-1) with an efficiency that was ∼4.6 × 103-fold less than that observed with MR. The inactivation arose predominantly from modifications at multiple sites and Tris-HCl, but not l-fuconate, afforded protection against inactivation. Similar to the reaction of 3-HP with MR, 3-HP modified the Brønsted base catalyst (Lys 220) at the active site of FucD, which was facilitated by the Brønsted acid catalyst His 351. Thus, the interactions of tartronate and 3-HP with MR and FucD revealed differences in binding affinity and reactivity that differentiated between the enzymes' enolization apparatuses.

Reprogramming biocatalytic futile cycles through computational engineering of stereochemical promiscuity to create an amine racemase.

Repurposing the intrinsic properties of natural enzymes can offer a viable solution to current synthetic challenges through the development of novel biocatalytic processes. Although amino acid racemases are ubiquitous in living organisms, an amine racemase (AR) has not yet been discovered despite its synthetic potential for producing chiral amines. Here, we report the creation of an AR based on the serendipitous discovery that amine transaminases (ATAs) can perform stereoinversion of 2-aminobutane. Kinetic modeling revealed that the unexpected off-pathway activity results from stereochemically promiscuous futile cycles due to incomplete stereoselectivity for 2-aminobutane. This finding motivated us to engineer an S-selective ATA through in silico alanine scanning and empirical combinatorial mutations, creating an AR with broad substrate specificity. The resulting AR, carrying double point mutations, enables the racemization of both enantiomers of diverse chiral amines in the presence of a cognate ketone. This strategy may be generally applicable to a wide range of transaminases, paving the way for the development of new-to-nature racemases.

Apolipoprotein A-I Binding Protein Inhibits the Formation of Infantile Hemangioma through Cholesterol-Regulated Hypoxia-Inducible Factor 1α Activation.

Infantile hemangioma (IH) is the most frequent vascular tumor of infancy with unclear pathogenesis; disordered angiogenesis is considered to be involved in its formation. Apolipoprotein A-I binding protein (AIBP)-also known as NAXE (NAD [P]HX epimerase)-a regulator of cholesterol metabolism, plays a critical role in the pathological angiogenesis of mammals. In this study, we found that AIBP had much lower expression levels in both tissues from patients with IH and hemangioma endothelial cells (HemECs) than in adjacent normal tissues and human dermal vascular endothelial cells, respectively. Knockout of NAXE by CRISPR-Cas9 in HemECs enhanced tube formation and migration, and NAXE overexpression impaired tube formation and migration of HemECs. Interestingly, AIBP suppressed the proliferation of HemECs in hypoxia. We then found that reduced expression of AIBP correlated with increased hypoxia-inducible factor 1α levels in tissues from patients with IH and HemECs. Further mechanistic investigation demonstrated that AIBP disrupted hypoxia-inducible factor 1α signaling through cholesterol metabolism under hypoxia. Notably, AIBP significantly inhibited the development of IH in immunodeficient mice. Furthermore, using the validated mouse endothelial cell (ie, EOMA cells) and Naxe-/- mouse models, we demonstrated that both endogenous AIBP from tumors and AIBP in the tumor microenvironment limit the formation of hemangioma. These findings suggested that AIBP was a player in the pathogenesis of IH and could be a potential pharmacological target for treating IH.

Sequence- and Structure-Based Mining of Thermostable D-Allulose 3-Epimerase and Computer-Guided Protein Engineering To Improve Enzyme Activity.

D-Allulose, a functional sweetener, can be synthesized from fructose using D-allulose 3-epimerase (DAEase). Nevertheless, a majority of the reported DAEases have inadequate stability under harsh industrial reaction conditions, which greatly limits their practical applications. In this study, big data mining combined with a computer-guided free energy calculation strategy was employed to discover a novel DAEase with excellent thermostability. Consensus sequence analysis of flexible regions and comparison of binding energies after substrate docking were performed using phylogeny-guided big data analyses. TtDAE from Thermogutta terrifontis was the most thermostable among 358 candidate enzymes, with a half-life of 32 h at 70 °C. Subsequently, structure-guided virtual screening and a customized strategy based on a combinatorial active-site saturation test/iterative saturation mutagenesis were utilized to engineer TtDAE. Finally, the catalytic activity of the M4 variant (P105A/L14C/T63G/I65A) was increased by 5.12-fold. Steered molecular dynamics simulations indicated that M4 had an enlarged substrate-binding pocket, which enhanced the fit between the enzyme and the substrate. The approach presented here, combining DAEases mining with further rational modification, provides guidance for obtaining promising catalysts for industrial-scale production.

Structural analysis of a bacterial UDP-sugar 2-epimerase reveals the active site architecture before and after catalysis.

The sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, was first identified ∼40 years ago in the O-antigen of Pseudomonas aeruginosa O:3,a,d. Since then, it has been observed on the O-antigens of various pathogenic Gram-negative bacteria including Bordetella pertussis, Escherichia albertii, and Pseudomonas mediterranea. Previous studies have established that five enzymes are required for its biosynthesis beginning with uridine dinucleotide (UDP)-N-acetyl-d-glucosamine (UDP-GlcNAc). The final step in the pathway is catalyzed by a 2-epimerase, which utilizes UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid as its substrate. Curious as to whether this biochemical pathway is found in extreme thermophiles, we examined the published genome sequence for Thermus thermophilus HB27 and identified five ORFs that could possibly encode for the required enzymes. The focus of this investigation is on the ORF WP_011172736, which we demonstrate encodes for a 2-epimerase. For this investigation, ten high resolution X-ray crystallographic structures were determined to resolutions of 2.3 Å or higher. The models have revealed the manner in which the 2-epimerase anchors its UDP-sugar substrate as well as its UDP-sugar product into the active site. In addition, this study reveals for the first time the manner in which any sugar 2-epimerase can simultaneously bind UDP-sugars in both the active site and the allosteric binding region. We have also demonstrated that the T. thermophilus enzyme is allosterically regulated by UDP-GlcNAc. Whereas the sugar 2-epimerases that function on UDP-GlcNAc have been the focus of past biochemical and structural analyses, this is the first detailed investigation of a 2-epimerase that specifically utilizes UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid as its substrate.

The Engineering, Expression, and Immobilization of Epimerases for D-allulose Production.

The rare sugar D-allulose is a potential replacement for sucrose with a wide range of health benefits. Conventional production involves the employment of the Izumoring strategy, which utilises D-allulose 3-epimerase (DAEase) or D-psicose 3-epimerase (DPEase) to convert D-fructose into D-allulose. Additionally, the process can also utilise D-tagatose 3-epimerase (DTEase). However, the process is not efficient due to the poor thermotolerance of the enzymes and low conversion rates between the sugars. This review describes three newly identified DAEases that possess desirable properties for the industrial-scale manufacturing of D-allulose. Other methods used to enhance process efficiency include the engineering of DAEases for improved thermotolerance or acid resistance, the utilization of Bacillus subtilis for the biosynthesis of D-allulose, and the immobilization of DAEases to enhance its activity, half-life, and stability. All these research advancements improve the yield of D-allulose, hence closing the gap between the small-scale production and industrial-scale manufacturing of D-allulose.

Peptide epimerase-dehydratase complex responsible for biosynthesis of the linaridin class ribosomal peptides.

Grisemycin, salinipeptin, and cypemycin belong to the linaridin class of ribosomally synthesized and posttranslationally modified peptides that contain multiple dehydrobutyrine and D-amino acid residues. The biosynthetic gene clusters of these linaridins lack obvious candidate genes for the dehydratase and epimerase required to introduce dehydrobutyrine and D-amino acid residues, respectively. However, we previously demonstrated that the grisemycin (grm) cluster contained cryptic dehydratase and epimerase genes by heterologous expression of this biosynthetic gene cluster in Streptomyces lividans and proposed that two genes (grmH and grmL) with unknown functions catalyze dehydration and epimerization reactions. In this study, we confirmed that both GrmH and GrmL, which were shown to constitute a protein complex by a co-purification experiment, were required to catalyze the dehydration, epimerization, and proteolytic cleavage of a precursor peptide GrmA by in vivo experiments. Furthermore, we demonstrated that GrmH/GrmL complex accepted salinipeptin and cypemycin precursor peptides, which possess three additional amino acids.

Structure of lasso peptide epimerase MslH reveals metal-dependent acid/base catalytic mechanism.

The lasso peptide MS-271 is a ribosomally synthesized and post-translationally modified peptide (RiPP) consisting of 21 amino acids with D-tryptophan at the C-terminus, and is derived from the precursor peptide MslA. MslH, encoded in the MS-271 biosynthetic gene cluster (msl), catalyzes the epimerization at the Cα center of the MslA C-terminal Trp21, leading to epi-MslA. The detailed catalytic process, including the catalytic site and cofactors, has remained enigmatic. Herein, based on X-ray crystallographic studies in association with MslA core peptide analogues, we show that MslH is a metallo-dependent peptide epimerase with a calcineurin-like fold. The crystal structure analysis, followed by site-directed mutagenesis, docking simulation, and ICP-MS studies demonstrate that MslH employs acid/base chemistry to facilitate the reversible epimerization of the C-terminal Trp21 of MslA, by utilizing two pairs of His/Asp catalytic residues that are electrostatically tethered to a six-coordination motif with a Ca(II) ion via water molecules.

A maize epimerase modulates cell wall synthesis and glycosylation during stomatal morphogenesis.

The unique dumbbell-shape of grass guard cells (GCs) is controlled by their cell walls which enable their rapid responses to the environment. The molecular mechanisms regulating the synthesis and assembly of GC walls are as yet unknown. Here we have identified BZU3, a maize gene encoding UDP-glucose 4-epimerase that regulates the supply of UDP-glucose during GC wall synthesis. The BZU3 mutation leads to significant decreases in cellular UDP-glucose levels. Immunofluorescence intensities reporting levels of cellulose and mixed-linkage glucans are reduced in the GCs, resulting in impaired local wall thickening. BZU3 also catalyzes the epimerization of UDP-N-acetylgalactosamine to UDP-N-acetylglucosamine, and the BZU3 mutation affects N-glycosylation of proteins that may be involved in cell wall synthesis and signaling. Our results suggest that the spatiotemporal modulation of BZU3 plays a dual role in controlling cell wall synthesis and glycosylation via controlling UDP-glucose/N-acetylglucosamine homeostasis during stomatal morphogenesis. These findings provide insights into the mechanisms controlling formation of the unique morphology of grass stomata.

Hepatic glucuronyl C5-epimerase combats obesity by stabilising GDF15.

BACKGROUND & AIMS: Disturbed hepatic metabolism frequently results in excessive lipid accumulation in the adipose tissue. However, the specific role of the liver-adipose axis in maintaining lipid homeostasis, as well as the underlying mechanism, has not yet been fully elucidated. In this study, we investigated the role of hepatic glucuronyl C5-epimerase (Glce) in the progression of obesity. METHODS: We determined the association between the expression of hepatic Glce and body mass index (BMI) in obese patients. Obesity models were established in hepatic Glce-knockout and wild-type mice fed a high-fat diet (HFD) to understand the effect of Glce on obesity development. The role of Glce in the progression of disrupted hepatokine secretion was examined via secretome analysis. RESULTS: Hepatic Glce expression was inversely correlated with BMI in obese patients. Moreover, Glce level was found to be decreased in the liver of a HFD murine model. Hepatic Glce deficiency led to impaired thermogenesis in adipose tissue and exacerbated HFD-induced obesity. Interestingly, decreased level of growth differentiation factor 15 (GDF15) was observed in the culture medium of Glce-knockout mouse hepatocytes. Treatment with recombinant GDF15 obstructed obesity progression derived from the absence of hepatic Glce, similar to the effect of Glce or its inactive mutant overexpressed both in vitro and in vivo. Furthermore, liver Glce deficiency led to diminished production and increased degradation of mature GDF15, resulting in reduced hepatic GDF15 secretion. CONCLUSIONS: Hepatic Glce deficiency facilitated obesity development, and decreased Glce expression further reduced hepatic secretion of GDF15, thereby perturbing lipid homeostasis in vivo. Therefore, the novel Glce-GDF15 axis plays an important role in maintaining energy balance and may act as a potential target for combating obesity. IMPACT AND IMPLICATIONS: Evidence suggests that GDF15 plays a key role in hepatic metabolism; however, the molecular mechanism for regulating its expression and secretion is largely unknown. Our work observes that hepatic Glce, as a key Golgi-localised epimerase, may work on the maturation and post-translational regulation of GDF15. Hepatic Glce deficiency reduces the production of mature GDF15 protein and facilitates its ubiquitination, resulting in the aggravation of obesity development. This study sheds light on the new function and mechanism of the Glce-GDF15 axis in lipid metabolism and provides a potential therapeutic target against obesity.

A Novel D-Psicose 3-Epimerase from Halophilic, Anaerobic Iocasia fonsfrigidae and Its Application in Coconut Water.

D-Psicose is a rare, low-calorie sugar that is found in limited quantities in national products. Recently, D-psicose has gained considerable attention due to its potential applications in the food, nutraceutical, and pharmaceutical industries. In this study, a novel D-psicose 3-epimerase (a group of ketose 3-epimerase) from an extremely halophilic, anaerobic bacterium, Iocasia fonsfrigidae strain SP3-1 (IfDPEase), was cloned, expressed in Escherichia coli, and characterized. Unlike other ketose 3-epimerase members, IfDPEase shows reversible epimerization only for D-fructose and D-psicose at the C-3 position but not for D-tagatose, most likely because the Gly218 and Cys6 at the substrate-binding subsites of IfDPEase, which are involved in interactions at the O-1 and O-6 positions of D-fructose, respectively, differ from those of other 3-epimerases. Under optimum conditions (5 µM IfDPEase, 1 mM Mn2+, 50 °C, and pH 7.5), 36.1% of D-psicose was obtained from 10 mg/mL D-fructose. The IfDPEase is highly active against D-fructose under NaCl concentrations of up to 500 mM, possibly due to the excessive negative charges of acidic amino acid residues (aspartic and glutamic acids), which are localized on the surface of the halophilic enzyme. These negative charges may protect the enzyme from Na+ ions from the environment and result in the lowest pI value compared to those of other 3-epimerase members. Moreover, without adjusting any ingredients, IfDPEase could improve coconut water quality by converting D-fructose into D-psicose with a yield of 26.8%. Therefore, IfDPEase is an attractive alternative to enhancing the quality of fructose-containing foods.

d-amino acid auxotrophic Escherichia coli strain for in vivo functional cloning of novel d-amino acid synthetic enzyme.

Various d-amino acids have been found in a wide range of organisms, including mammals. Although the physiological functions of various d-amino acids have been reported or suggested, the molecular basis of these biological functions has been elucidated in only a few cases. The identification of a d-amino acid biosynthetic enzyme is a critical step in understanding the mechanism of the physiological functions of d-amino acids. While in vivo functional screening can be a powerful tool for identifying novel metabolic enzymes, none of the existing organisms exhibit growth dependent on d-amino acid other than d-Ala and d-Glu. Here, we report the first organism that exhibits non-canonical d-amino acid auxotrophy. We found that an Escherichia coli strain lacking the major d-Ala and d-Glu biosynthetic enzymes, alr, dadX, and murI, and expressing the mutated d-amino acid transaminase (DAAT) gene from Bacillus sp. YM-1 (MB3000/mdaat+ ) grew well when supplemented with certain d-amino acid. A multicopy suppression study with plasmids encoding one of the 51 PLP-dependent enzymes of E. coli showed that MB3000/mdaat+ could detect weak and moonlighting racemase activity, such from cystathionine ß-lyase (MetC) and a negative regulator of MalT activity/cystathionine ß-lyase (MalY)-these exhibit only a few tenths to a few thousandths of the racemization activity of canonical amino acid racemases. We believe that this unique platform will contribute to further research in this field by identifying novel d-amino acid-metabolizing enzymes.

Involvement of the Streptococcus mutans PgfE and GalE 4-epimerases in protein glycosylation, carbon metabolism, and cell division.

Streptococcus mutans is a key pathogen associated with dental caries and is often implicated in infective endocarditis. This organism forms robust biofilms on tooth surfaces and can use collagen-binding proteins (CBPs) to efficiently colonize collagenous substrates, including dentin and heart valves. One of the best characterized CBPs of S. mutans is Cnm, which contributes to adhesion and invasion of oral epithelial and heart endothelial cells. These virulence properties were subsequently linked to post-translational modification (PTM) of the Cnm threonine-rich repeat region by the Pgf glycosylation machinery, which consists of 4 enzymes: PgfS, PgfM1, PgfE, and PgfM2. Inactivation of the S. mutans pgf genes leads to decreased collagen binding, reduced invasion of human coronary artery endothelial cells, and attenuated virulence in the Galleria mellonella invertebrate model. The present study aimed to better understand Cnm glycosylation and characterize the predicted 4-epimerase, PgfE. Using a truncated Cnm variant containing only 2 threonine-rich repeats, mass spectrometric analysis revealed extensive glycosylation with HexNAc2. Compositional analysis, complemented with lectin blotting, identified the HexNAc2 moieties as GlcNAc and GalNAc. Comparison of PgfE with the other S. mutans 4-epimerase GalE through structural modeling, nuclear magnetic resonance, and capillary electrophoresis demonstrated that GalE is a UDP-Glc-4-epimerase, while PgfE is a GlcNAc-4-epimerase. While PgfE exclusively participates in protein O-glycosylation, we found that GalE affects galactose metabolism and cell division. This study further emphasizes the importance of O-linked protein glycosylation and carbohydrate metabolism in S. mutans and identifies the PTM modifications of the key CBP, Cnm.

Enzymatic C4-Epimerization of UDP-Glucuronic Acid: Precisely Steered Rotation of a Transient 4-Keto Intermediate for an Inverted Reaction without Decarboxylation.

UDP-glucuronic acid (UDP-GlcA) 4-epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4-oxidation of the substrate by NAD+ and rotation of a decarboxylation-prone ß-keto acid intermediate in the active site, enabling stereoinverting reduction of the keto group by NADH. We reveal the elusive rotational landscape of the 4-keto intermediate. Distortion of the sugar ring into boat conformations induces torsional mobility in the enzyme's binding pocket. The rotational endpoints show that the 4-keto sugar has an undistorted 4 C1 chair conformation. The equatorially placed carboxylate group disfavors decarboxylation of the 4-keto sugar. Epimerase variants lead to decarboxylation upon removal of the binding interactions with the carboxylate group in the opposite rotational isomer of the substrate. Substitutions R185A/D convert the epimerase into UDP-xylose synthases that decarboxylate UDP-GlcA in stereospecific, configuration-retaining reactions.

Bifunctional Epimerase/Reductase Enzymes Facilitate the Modulation of 6-Deoxy-Heptoses Found in the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni is a human pathogen and the leading cause of food poisoning in the United States and Europe. Surrounding the exterior surface of this bacterium is a capsular polysaccharide (CPS) that consists of a repeating sequence of common and unusual carbohydrate segments. At least 10 different heptose sugars have thus far been identified in the various strains of C. jejuni. The accepted biosynthetic pathway for the construction of the 6-deoxy-heptoses begins with the 4,6-dehydration of GDP-d-glycero-d-manno-heptose by a dehydratase, followed by an epimerase that racemizes C3 and/or C5 of the product GDP-6-deoxy-4-keto-d-lyxo-heptose. In the final step, a C4-reductase catalyzes the NADPH reduction of the resulting 4-keto product. However, in some strains and serotypes of C. jejuni, there are two separate C4-reductases with different product specificities in the gene cluster for CPS formation. Five pairs of these tandem C4-reductases were isolated, and the catalytic properties were ascertained. In four out of five cases, one of the two C4-reductases is able to catalyze the isomerization of C3 and C5 of GDP-6-deoxy-4-keto-d-lyxo-heptose, in addition to the catalysis of the reduction of C4, thus bypassing the requirement for a separate C3/C5-isomerase. In each case, the 3'-end of the gene for the first C4-reductase contains a poly-G tract of 8-10 guanine residues that may be used to control the expression and/or catalytic activity of either C4-reductase. The three-dimensional structure of the C4-reductase from serotype HS:15, which only does a reduction of C4, was determined to 1.45 Å resolution in the presence of NADPH and GDP.

Endogenous d-serine exists in the mammalian brain independent of synthesis by serine racemase.

Activation of N-methyl-d-aspartate receptors (NMDARs) requires binding of a co-agonist in addition to l-glutamate. d-serine binds to the co-agonist site on GluN1 subunits of NMDARs and modulates glutamatergic neurotransmission. While loss of GluN1 subunits in mice results in neonatal death due to respiratory failure, animals that lack a d-serine synthetic enzyme, serine racemase (SR), show grossly normal growth. However, SR-independent origins of d-serine in the brain remain unclarified. In the present study, we investigated the origin of brain d-serine in mice. Loss of SR significantly reduced d-serine in the cerebral cortex, but a portion of d-serine remained in both neonates and adults. Although d-serine was also produced by intestinal bacteria, germ-free experiments did not influence d-serine levels in the cerebral cortex. In addition, treatment of SR-knockout mice with antibiotics showed a significant reduction of intestinal d-serine, but no reduction in the brain. On the other hand, restriction of dietary intake reduced systemic circulation of d-serine and resulted in a slight decrease of d-serine in the cerebral cortex, but did not account for brain d-serine found in the SR-knockout mice. Therefore, our findings show that endogenous d-serine of non-SR origin exists in the brain. Such previously unrecognized, SR-independent, endogenous d-serine may contribute baseline activity of NMDARs, especially in developing brain, which has minimal SR expression.

Reshaping the Binding Pocket of Cellobiose 2-Epimerase for Improved Substrate Affinity and Isomerization Activity for Enabling Green Synthesis of Lactulose.

Enzymatic isomerization of lactose into lactulose via cellobiose 2-epimerase (CE) could provide an eco-friendly route for the industrial production of lactulose, a valuable food prebiotic. However, poor substrate affinity for lactose and preference for epimerization over isomerization hinder this application. Previous studies on CE improvement have focused on random mutagenesis or active site rational design; little is known about the relationship between substrate binding and enzyme efficacy, which was hence the subject of this study. First, residues 372W and 308W were identified as key for disaccharide recognition in CEs based on crystal structure alignment of the N-acetyl-glucosamine 2-epimerase superfamily and site-directed mutation. This binding domain was then reshaped through site saturation mutagenesis, resulting in seven mutants with enhanced isomerization activity. The optimal mutant CsCE/Q371E had significantly enhanced substrate affinity (Km, 269.65 mM vs Km, 417.5 mM), reduced epimerization activity, and 3.3-fold increased isomerization activity over the original CsCE. Molecular dynamics simulation further revealed that substituting Gln-371 with Glu strengthened the hydrogen-bonding network and altered the active site-substrate interactions, increasing the substrate stability and shifting the catalytic direction. This study uncovered new information about the substrate binding region and its mechanisms and impact on CE catalytic performance, paving the way for potential commercial applications.